Recombinant cellular expression systems for the production of proteins are known. These systems range from bacteria, yeast and fungi to plant cells, and from insect cells to mammalian cells. The choice for the production host and expression system generally depends on considerations such as the ease of use, cost of culturing, growth characteristics, production levels and the ability to grow on serum-free medium. It is known that the cellular expression systems mentioned above also differ in the capacity to exert co- and post-translation modifications such as folding, phosphorylation, γ-carboxylation, and γ-hydroxylation. Despite the recognition that the choice of Despite the recognition that the choice of the recombinant expression system may have dramatic consequences on the ultimate structure of the expressed proteins, post-translational modifications have in general not played a decisive role in selecting a suitable expression system for a given protein.
In the last number of years, studies have revealed more about the complexities of differential post-translational modifications of human proteins and the potential implications on functions in the human body. For example, relatively recent findings suggest that differential glycosylation patterns of human proteins that occur in the blood (so-called ‘serum-type’ modifications) are different from the ones that occur in the cerebrospinal fluid in the brain (‘brain-type’ modifications). This difference may be a key issue that is of paramount importance for the design of effective therapeutics.
In general, human neural glycoproteins are characterized by their glycosylation, which has been referred to in literature as ‘brain-type’ glycosylation (Margolis and Margolis 1989; Hoffmann et al. 1994). In contrast to ‘serum-type’ glycosylated proteins (i.e., glycoproteins circulating in the blood) brain-type glycosylated proteins characteristically possess complex-type N-linked sugars that are modified with α1,3-linked fucose attached to N-acetyl-glucosamine in lactosamine-type antennae thereby forming Lewis x or sialyl-Lewis x structures (FIG. 5). There are two types of Lewis x structures: One with a terminal galactose residue and one with a terminal N-acetyl-galactosamine (GalNac) residue. If these terminal groups are linked to a sialic acid, the Lewis x structure is called a sialyl Lewis x structure. Another difference between serum-type and brain-type oligosaccharides is that the latter often contain terminal N-acetyl-glucosamine and/or terminal galactose, and may include a terminal N-acetyl-galactosamine modification, whereas serum-type oligosaccharides usually contain only low amounts of such structures.
Oligosaccharides that are generally found on proteins circulating in the serum often contain heavily galactosylated structures. This means that a galactose is linked to a peripheral N-acetyl-glucosamine thereby forming a lactosamine structure. The glycoprotein is in this way protected from endocytosis by the N-acetyl-glucosamine receptors (i.e., receptors that recognize terminal N-acetyl-glucosamine) present in hepatic reticuloendothelial cells and macrophages (Anchord et al. 1978; Stahl et al. 1978). Serum-type oligosaccharides usually also contain terminal sialic acids (also often referred to as neuraminic acid) which protect the glycoprotein from clearance through the asialoglycoprotein receptor. These clearance mechanisms specifically apply to glycoproteins circulating in the blood and are probably lacking in the human central nervous system (CNS) (Hoffmann et al. 1994).
Recombinant expression systems for the production of proteins comprising ‘serum-type’ modifications are available in the art, as exemplified by Chinese Hamster Ovary (CHO) cells and Baby Hamster Kidney (BHK) cells. For the production of proteins with other modifications, such as ‘brain-type’ modifications however, no such convenient systems have been described. Hence, there is a need for expression systems that take into account the different post-translational modifications on therapeutic proteins. In particular, a need exists for an efficient expression system for proteins comprising ‘brain-type’ post-translational modifications.
Proteins that have these specific needs may be beneficial in the treatment of all sorts of disorders, among which are the diseases related to the CNS, the peripheral nervous system and heart tissue. Disorders affecting the CNS encompass different kinds of afflictions such as acute brain damage, neurodegenerative diseases and other dysfunctions such as epilepsy, schizophrenia and mood disorders. Other pathological disorders that might afflict neural cells and tissues are due to injuries that might be a result of hypoxia, seizure disorders, neurotoxin poisoning, multiple sclerosis, hypotension, cardiac arrest, radiation or hypoglycemia. Neural injuries might also occur during surgical procedures such as aneurysm repair or tumor resection.
An example of a protein having different roles which are at least in part related to differences in post-translational modifications, is a hormone known as erythropoietin (EPO). EPO, a protein famous for its role in differentiating hematopoietic stem cells into red blood cells, has several other functions, including functions in neural tissues. A role of EPO in the development of the CNS has been suggested (Dame et al. 2001). EPO protein has also been detected in the cerebrospinal fluid (CSF) of human neonates and adults (Juul et al. 1997; Buemi et al. 2000). EPO as present in the CSF appears to be produced locally in the brain as it does not cross the intact blood-brain barrier (Marti et al. 1997; Buemi et al. 2000). The regulation of EPO expression is tissue-specific, which further strengthens the hypothesis that EPO has tissue-specific functions that are different in the brain and the bone marrow (Masuda et al. 1999; Chikuma et al. 2000; Sasaki et al. 2001). It has therefore been postulated that EPO, in addition to its heamatopoietic function, may have a neurotrophic role. Neurotrophic factors are defined as humoral molecules acting on neurons to influence their development, differentiation, maintenance, and regeneration (Konishi et al. 1993). The results of several studies have now demonstrated that EPO can act as a neurotrophic factor (e.g. Sadamoto et al. 1998; Brines et al. 2000). In addition to the mentioned effects of EPO on erythropoiesis and neuroprotection, other roles of EPO have been described, e.g. in endothelial cells and muscle cells. It has been well established in the art that the effect of (recombinant) EPO depends heavily on the glycosylation pattern of the oligosaccharides present on the protein. The N-linked oligosaccharides of human EPO are highly important for its well-known biological activity: the stimulation of erythropoiesis (Takeuchi and Kobata 1991; Wasley et al. 1991; Tsuda et al. 1990; Morimoto et al. 1996; Takeuchi et al. 1989; Misaizu et al. 1995).
In the case of EPO, one can also refer to a serum-type EPO (or a ‘renal-type’, or a ‘urinary-type’ EPO) for the protein that is produced in the kidney and that circulates in the blood, as compared to EPO that is been produced by other tissues such as the brain (brain-type). Production and purification systems for serum-type EPO are well established in the art, and recombinantly produced serum-type EPO is routinely and successfully used for instance in patients suffering from a low red blood cell level. It is well established in the art that this recombinant EPO had to fulfill all requirements of a stable protein that could circulate in the bloodstream for a sufficient amount of time to enable the induction of erythropoiesis. Usually a CHO or BHK based cell system is used for the production of EPO with these characteristics. However, the serum-type EPO resulting from this production and purification system is relatively useless in the treatment of disorders related to the Central- or Peripheral Nervous system as well as in the treatment of afflictions related to ischemia/reperfusion induced disorders. This is because of its glycosylation pattern that is not suited for the treatment of these disorders, and also because it leads to an increase in the number of red blood cells (erythropoiesis) due to its strong hematopoietic activity, which is to be qualified as undesirable side effects in the context of these non-hematopoietic disorders (Wiessner et al, 2001). Hence, a need exists for new production systems for proteins such as EPO, that have the characteristic features of an EPO molecule that is active in the brain or in tissues that involve selectin-based transport or targeting. Furthermore, a need exists for pharmaceutically acceptable preparations of proteins such as EPO, with post-translational modifications that differ from the serum type glycosylation, preferably having a brain-type glycosylation, and efficient production and purification systems to provide for these.
Another example of a protein that has different glycosylation patterns in separate tissues, suggesting a differential role of the different glycosylation patterns, is transferrin, which occurs in significant amounts as asialotransferrin in the CSF but not in that form in serum (Van Eijk et al. 1983; Hoffmann et al. 1995).
A certain family of glycoproteins, named selectins, play an important role in the initial steps of adhesion of leukocytes to the endothelium in ischemia/reperfusion injury. There are three members in the selectin family: P-selectin, E-selectin and L-selectin. Selectins have a lectin domain that recognizes the sugar structures of the glycoprotein ligands binding to them. There is a possible role for the sialyl Lewis x modifications in oligosaccharides in binding to selectins (Foxall et al. 1992). Several studies have indicated the importance of selectins and sialyl Lewis x structures for the adhesion of leukocytes in models of ischemia/reperfusion. The sialyl Lewis x oligosaccharide Slex-OS was for instance shown to be cardioprotective in a feline model of ischemia/reperfusion by reducing cardiac necrosis by 83% (Buerke et al. 1994). Furthermore, patent application WO 02/38168 describes the use of selectin binding proteins comprising sialyl Lewis x structures for use as anti-inflammatory agents in the treatment of various diseases. However, suitable expression systems for the preparation of proteins comprising (sialyl) Lewis x glycans have not been described. Hence, a need exists for a recombinant expression system for proteins in need of predetermined glycosylation structures, such as (sialyl) Lewis x structures. More in general, there is a need for expression systems for recombinant production of proteins in need of predetermined post-translational modifications.
    A.) PER.C6™-EPO incubated with PNGase F and neuraminidase.    B.) PER.C6™-EPO incubated with PNGase F, neuraminidase and galactosidase.    C.) PER.C6™-EPO incubated with PNGase F and neuraminidase, and subsequently treated with galactosidase and bovine kidney fucosidase.    D.) PER.C6™-EPO incubated with PNGase F and neuraminidase, and subsequently treated with galactosidase, bovine kidney fucosidase and GlcNAc-ase.    E.) PER.C6™-EPO incubated with PNGase F and neuraminidase, and subsequently treated with galactosidase and almond meal fucosidase.    F.) PER.C6™-EPO incubated with PNGase F and neuraminidase, and subsequently treated with galactosidase, almond meal fucosidase and GlcNAc-ase.